1. Field of the Invention
The present invention relates to an animal cell line which is capable of growing in a substantially protein-free medium and has a substance capable of enhancing replication of exogenous genes, a transformant obtained by transfecting said animal cell line with a recombinant DNA containing at least one exogenous gene capable of expression, a method of producing exogenous gene products by using the transformant, a method of using the animal cell line for preparing said transformant and a method of using said transformant for producing the exogenous gene products.
2. Description of the Prior Art
Recombinant DNA procedures have been widely used which comprise the steps of transfecting a DNA containing a specific gene(s) into a cultured cell, culturing the cell to express the gene products and obtaining the products.
Most of cultured animal cells useful for recombinant DNA procedures are cultured in a medium containing natural products of which the chemical composition is unknown, such as serum. For example, it is known that COS-1 cell line which was modified to produce mouse interferon .gamma. is cultured in serum-containing Dulbecco's modified minimum essential medium (P. W. Gray, D.V. Goeddel., Proc. Natl, Acad. Sci. U.S.A., 80: 5842-5846, 1983). Thus, great efforts are required to remove impurities when collecting and purifying gene products. At the present time, there have been used processes wherein a cell transfected to express gene products is generally cultured first in a serum-containing medium and then in a serum-free medium to thereby easily collect and purify the gene products However, when a serum-containing medium is changed to a serum-free medium, the proliferation potency of the cultured cell decreases rapidly. In order to prevent this decrease, one needs to add proteins, such as insulin, transferrin, albumin and the like, to the serum-free medium. Addition of these proteins increases the cost of the medium because these proteins are more difficult to purify and more expensive than other low molecular components of the medium.
Vero-317 cell line derived from African green monkey kidney, L-P3 cell line derived from mouse and HeLa-P3 cell line derived from human cervix carcinoma and the like are known as animal cell lines which can grow in protein-free media. However, it has not been reported that these cells amplify exogenous genes extrachromosomally.
Some virus DNAs which can be used as vectors in recombinant DNA procedures are amplified in a host cell without being integrated in the nuclear chromosome of the host cell. For example, when a double-stranded circular DNA containing an origin of DNA replication of SV40 virus is transfected into a monkey cell having a large T antigen derived from SV40 virus, the DNA is replicated and amplified in the host monkey cell without being integrated in the nuclear chromosome of the host monkey cell (edited by Uchida, Oishi, Furusawa., "Use and Practice Mannual of Animal Cells"., PP 388-393, L.I.C.Co., 1984). Further, when a Baculo virus DNA is transfected into an insect cell, the DNA is replicated and amplified in the host insect cell without being integrated in the nuclear chromosome of the host insect cell (Maeda., Experimental Medicine., vol. 7, No. 13, 146-151, 1989; Sekine et al., Gene., vol. 65, 187, 1988; and Miyajima et al., Gene., Vol. 58, 273, 1987).
So far, an animal cell line has not been known which can grow in a substantially protein-free medium and can amplify transfected genes extrachromosomally.